Several scientific publications were published about compounds of antiplasmid effect. There was no mention about antiplasmid effect of hydroxy-benzoic acids up to now in the literature, only some publications dealing with their antibacterial effect appeared in this field (e.g. Himejima, Masaki; Kubo, Isako, J. Agric. 1991, 39/2, 411-421.)
The importance of compounds of antiplasmid effect consists of their capability to destroy the acquired resistance of bacteria against antibiotics which can often save lives. The effect of bacteria living in the environment to the health seems unimportant for a human being while he or she is healthy. It becomes important only, when he or she gets ill because of a bacterial infection. The major part of these bacterial illnesses can be cured by means of antibiotics, but there is a growing number of infections, that are fatal because of the antibiotic resistance of the disease germ.
The phenomenon of insensibility to the antibiotics, the resistance spreads not only to human pathogenic germs, but also to pathogenic germs of animals and plants as well.
The resistance occurs among both the aerobic and anaerobic bacteria. These strains of bacteria are resistant to a multitude of antibiotics, even to those, which are reserved for the treatment of heavy infections, e.g. aminoglycosides, types of penicillin, cephalosporine and tetracycline,
We set the target to produce one or more compounds with antiplasmid effect. We have found that certain hydroxy-benzoic acid derivatives have antiplasmid effect. There are some specific sorts of bacteria causing often epidemic infections, which are capable to inactivate several antibiotics.
The bacteria can be classified into three groups according to the origin of their resistance to antibiotics
a) resistance pertaining to the species, which is a well-known phenomenon of some species of bacteria to some kinds of antibiotics,
b) resistance, which originates from a mutation, a phenomenon of small probability with regard to a kind of antibiotic, it has a small practical importance by this reason,
c) The most dangerous kind of resistance, the so-called plasmid-coupled infectious resistance is in the focus of interest, which has epidemic growth among the bacteria.
The plasmids are DNA molecules, which have special proliferation mechanism and carry genetic information independently from the chromosomes of bacteria and they are capable to transmit various properties, e.g. resistance to antibiotics, virulence and other ones.
The plasmid of resistance enables the proliferation of bacteria in the presence of an antibiotic and the bacteria can transmit this feature not only to their descendants but even to other bacteria in their environment. Such epidemic spreading of resistance to antibiotics in the bacterial flora of humans and animals is well-known since about 30 years.
The antagonistic effect to replication of plasmids of acridine paints, the ethidium bromide and the sodium dodecil sulphate was discovered by former research works, these compounds are not eligible for medical purposes because of their high toxicity. The recognition, that two well-known medicaments, chloro-promazine and prometazine eliminated the resistance to tetracycline, chloro-amphenicol and sulfonamide at 10 to 30% of bacteria culture Escherichia coli in vitro, underlay to the experiments aiming elimination of plasmids. The experiments were done with several well-known, in the literature described strains of bacteria cultures and cell cultures with loss of specific plasmids on specific selective culture media.
The elimination of R-plasmids and Fxe2x80x2lac plasmids, as well as inhibition of transmission of R-plasmid transfer were studied.
The more and more extending resistance causes the research work in the field of medicaments to search continuously for new antibiotics and to supply the medicine with them.
The usage of compounds being subject of our application could result not only in a great economic benefit, but a great number of people could be successfully cured, who are actually beyond recovery because of the resistance, provided the in vivo experiments give similar results.
We found three compounds effective from 36 ones which were studied by in vitro experiments for antiplasmid effect. These are the compounds under mark FC-1181, FC-1186 and Sila-439. The compound sila-429 (75%) was compared against three representatively antibiotic resistant strains of bacteria, as follows: the polyresistant Acinetobacter anitratus, the resistant to gentamycine staphylococcus aureus and the Methycilline resistant Staphylococcus aureus strains.
The fact, that 3 compounds from 36 ones proved to be active in the searched effect, should be considered as an outstanding result even at international level.
We performed a preliminary study of toxicology, and established, that the mice inoculated with a dose of 50 mg/kg lived and moved vividly exactly like those in the control group, which were inoculated intraperitoneally with 0.2 ml of physiologic saline solution
We used the usual dose of inoculation i.e. 0.2 ml per 10 g weight. When we augmented the dose ten times to 500 mg/kg, spasms occurred in the 5 inoculated mice in two minutes after the intraperitoneal inoculation and all the five mice died.
It can be supposed on the base of preliminary toxicological experiment that the value LD50 is 4 to 6 times higher than the effective in vitro dose.
This latter fact should be studied in an institute dealing with toxicology and clinical pharmacology.
The experiments should be completed with examinations for plasmid elimination in an in vivo environment.
The results of experiments and calculations of quantum chemistry in relation to the examined molecules encouraged us to indicate some perspectives to design antiplasmid medicaments on the base of the relationship existing between the biological effects and the structures of molecules.
Certain compounds appeared to be promising on the base of the results of preliminary experiments and calculations. Some representatives of the mentioned groups of compounds were synthesised and examined in laboratory experiments by this reason.
The Hungarian patent specification under No. HU 180,334 (IPC C07C 87/78) describes production of 1,1-biphenyl-2-il-alkyl-amin derivatives.
The U.S. patent specification of U.S. Pat. No. 4,695,631 (IPC C07C 87/24) describes a method for producing enamines or -imines.
Antibiotics are often used to eliminate plasmids or other elements similar to chromosomes from the host cells. Michel, Briand and his fellow workers describe usage of several 4-quinolone derivatives (J. Antimicrob. Chemother., 18, 667-674 /1986/) included cinoxacine and novobiocine with the aim to extirpate various plasmids of resistance to medicaments from the cells of the family Enterobacteriaceae.
A special plasmid development method, a map of plasmid restriction and functions, as well as a flowchart of said method are described for producing somatotropic hormone for cattle in the US document laid open to public inspection of No. 54415/1990.
It can be established on the base of the cited sources of literature that no relevant solution was published, which affects the subject matter of the invention, they cannot be reviewed by his reason.
We tested a multitude of representatives of the group of compounds that we supposed to have antiplasmid effect, as we endeavoured to search for base of following research activity (that should be the target of the invention) to approach our aims in our targeted work but having elements of fundamental research, by means of analysis of the relationship between the chemical structure and biological effect.
We could establish a well recognisable relation to the antiplasmid effect depending on the substituents and functional groups.
We established during our experiments, that the derivatives of benzoic acid and those of benzoic acid imides having the general formula (I) 
as new compounds have antiplasmid effect. The general formula (I) is shown in the FIG. 1.
The substituents of the compound are as follows:
Q means: xcex2-picolinyl-methoiodide or 1-methyl-4-piperidyl or
3-(4xe2x80x2-fluorophenyl)-2,2-dimethyl-2-sila-propyloxy, or
2-dimethyl-amino-ethyl-methoiodide group and
Y means: 2,2-dimethyl-2-sila-hexyloxy or
3-(4xe2x80x2-fluorophenyl)-2,2-dimethyl-2-sila-propyloxy or
5-(4xe2x80x2-fluorophenyl)-4,4-dimethyl-4-sila-pentyloxy or
3-trimethylsilil-propyloxy group.
The scope of the present invention includes the pharmaceutical compositions with antiplasmid effect and the method of their production. We performed a search for previous publications in the Chemical Abstracts under keywords xe2x80x9cBenzamidexe2x80x9d and xe2x80x9cBenzoic Acidxe2x80x9d from 1962 to June 1998 and among HU, DE, EP and US patent specifications according to classification codes specified in the application form. No anteriority was found by the novelty search where would be found the describing of the derivatives of benzoic acid and benzoic acid amides of the general formula I with the substituents as defined above. The compounds and the method of their production, as well as their application as active substance of pharmaceutical compositions are new by this reason.
The compounds described in the literature and showing only similarity to our ones deviate of the general formula (1). Such compounds are the o-dimethyl-methoxy-silil-benzoic acid methylesher and the o-trimethoxy-silil-benzoic acid trimethyl-silil-ester There-is an oxo-oxygen atom in the alkylenic chain in the compounds which contain alkylene-oxy connection. Such compound is the 2-[2-/4-morpholinyl/-2-oxo-ethoxy]-benzoic acid-amid The 3-[2-n-butyl-1-{/trimethyl-silil-/ethoxy-methyl}-1H-imidazol-5-il]-benzoic acid-methyl ester differs from the compounds of the general formula (I) by the ethoxy-methyl-imidazolyl attachment between the silicon atom and the basic frame. The 2-formyl-5-methoxy-6-methyl-3-[dimethyl-/1,1,2-trimethyl-propyl/-silanol-oxy]-benzoic acid-allyl ester differs by its polysubstituted ring from the compounds required by the present application. The defined compounds show considerable antiplasmid effect in experiments in vitro. The compounds Sila-439 (75%9 and FC-2006/C have among others outstanding plasmid eliminating ability (in concentration of 50 xcexcg/ml). The compounds characterised by the general formula (I) are not only new, but they are unknown and not described in the literature, as there is not even any reference to them in the literature. The compound Sila-439 i.e. 4-(3-trimethyl-silil-propyloxy)-benzoic acid-2-dimethyl-amino-ethyl-ester-methoiodide has an extraordinary good eliminating ability The compounds marked by FC 2006/C and FC 2012 have good eliminating ability, as well.
The compound FC 2006/C is 4(dimethyl-n-butyl-silil-methyloxy)-benzoic acid-(3-pyridyl)-methylester-methoiodide.
The compound FC 2012 is 4-(dimethyl-n-butyl-silil-methyloxy)-benzoic acid-3-dimethylamino-propyl-ester-methoiodide.
The importance of these compounds is that they are capable to eliminate the acquired resistance of bacteria against antibiotics, and the said elimination can save lives.
On the one hand the compounds of the invention are capable to eliminate the plasma which causes the resistance; on the other hand they can hinder, that the plasma of resistance will be transmitted from the bacteria of xe2x80x9cinfectiousxe2x80x9d to the xe2x80x9chealthyxe2x80x9d bacteria and an xe2x80x9cepidemicxe2x80x9d would be created.
The compound Sila-439 is suitable to registration as medicament because its eliminating effect is more extended.
The eliminating effect of the compound FC-2006/C is almost 100% but only to one E. coli plasmid.
Methods Used:
The bacteriostatic effect was inspected on a liquid culture medium using method of serial dilution. We inoculated 0.05 ml of culture of the examined bacteria being in exponential phase (i.e. of optical density 0.4) in dilution by 10xe2x88x924 into broth culture medium MTE of 5 ml. The culture of E. coli of optical density 0.4 contained 4.1 to 5.2xc3x97108, the one of Staphylococcus aureus of similar density contained 2.2 to 8.2xc3x97107 settlement building units per ml. The culture medium units infected by the same number of bacteria and containing different quantities of compounds (0 to 30xc3x9710xe2x88x924 M) being examined were kept at the temperature 37xc2x0 C. during 18 hours. We examined thereafter the smallest concentration of each compound, which still inhibits the proliferation of the identical number of bacteria. The examinations for the antiplasmid effect could be begun after having been obtained the mentioned date, because these effects can be detected only at values lower than MIC.
The Elimination of R-Plasmid:
We made a dilution of 10xe2x88x924 from a preliminary culture of 16 hours of E. coli strains containing R-factor, and dosed 0.05 ml therefrom into a broth culture medium MTE of 5 ml. We completed the cultures with the examined compounds in different concentrations and kept them at the temperature 37xc2x0 C. during 24 to 48 hours without aeration. We made different dilutions from the cultures and streaked 0.1 ml of them onto EP agar plates. Having incubated them during the night we made a replication from the plates containing the cultures onto plates containing the corresponding antibiotics.
The plates containing antibiotics were incubated at 37xc2x0 C. during 24 hours then the settlements of bacteria on them were compared to those on the master plates and the requirement of nutrition of the bacteria was examined.
The Elimination of Fxe2x80x2lac Plasmid
We made a dilution of 10xe2x88x924 from a culture of 16 hours of the bacteria strain K12 LE140 of E. coli and we inoculated 0.05 ml (1 to 5xc3x97103 cells) of it into 5 ml of broth culture medium MTE. The cultures with plasmid eliminating compounds added in different concentrations were incubated at 37xc2x0 C. during 24 hours. We made dilutions from the contents of tubes, in which growth of bacteria were detected, then 0.1 ml of these dilutions were streaked onto EMB agar. The plates were incubated at 37xc2x0 C. during 24 hours, then we counted the settlements of bacteria of types lac+ and lacxe2x88x92, which provides basis to calculate the percent of inhibition.
The Inhibition of R-Plasmid Transfer
We made tenfold dilutions of fresh donor culture of E. coli C600 R144 drd-3 and of fresh recipient culture resistant to ir and in some cases to nalid-x acid of E. coli K12 W1 at optical density of 0.4 (60 nm), then we mixed together 1.0 ml of donor culture and 1.0 ml of recipient culture and kept the samples at the temperature 37xc2x0 C. 0 to 24 hours in presence of the applied compounds.
As control the mixture of donor and recipient cultures of the same proportion was incubated.
We performed experiments of two types to clarify the manner of action. The donor cells were previously treated during 5 minutes with the compound to be examined being in corresponding concentration, and the recipient cells were added thereafter in the experiments of one type The compounds to be examined were added 5 minutes after mixing together the donor and recipient cultures in the experiments of the other type. The target of our examinations was the inhibition of the conjugated transfer at the formed pairs. We made tenfold dilutions of the samples with physiological saline solution, the formed pairs of cells were divided by shaking during 1 minute then we made following dilutions where no more resistance transfer passed (see. Beverley and fellow-workers, 1981).
We streaked quantities of 0.1 ml form the dilutions onto MTE agar plates containing 50 xcexcg/ml of kanamycine and onto plates containing 500 xcexcg/ml of sodium azide to determine the number of recombinants. The number of donor bacteria was established only on the culture medium containing kanamycine, the number of recipient ones was established only on the culture medium containing sodium azide. The plates were kept during 48 hours at 37xc2x0 C.
The more and more extending resistance causes the research work in the field of medicaments to make enormous investments to search continuously for new antibiotics and to supply the medicine with them.
We performed preliminary examinations of toxicity of the compound Sila-439 having very good plasmid elimination effect (the 7.0 g of material which was handed), because it has not only the above mentioned effect, but a considerable antibacterial one, too.
The previously established MIC (minimal inhibiting concentration) values are as follows:
for Escherichia coli (resistant to Chlorocid) 70 xcexcg/ml,
for Acinetobactes anitratus (resistant to Chlorocid) 80 xcexcg/ml,
for Staphylococcus aureus (resistant to gentamycine) 180xcexc/ml.
The antiplasmid effect manifests at the concentration 40 xcexcg/ml.
We performed preliminary studies of toxicity and established, that the mice inoculated with a dose of 50 mg/kg lived and moved vividly exactly like those in the control group, which were inoculated intraperitoneally with 0,2 ml of physiologic saline solution.
We used the usual dose of inoculation i.e. 0,2 ml per 10 g weight. When we augmented the dose ten times to 500 mg/kg, spasms occurred in the 5 inoculated mice in two minutes after the intraperitoneal inoculation and all the five mice died. It can be supposed on the base of preliminary in vivo toxicological experiment that the value LD50 is 4 to 6 times higher than the effective in vitro dose. The exact definition of this value should be done in an institute dealing with toxicology and clinical pharmacology.
The usage of compounds being subject matter of our application could result not only in a great economic benefit, but a great number of people could be successfully cured, who are actually beyond recovery because of the resistance.
The subject matters of the invention are as follows.
A compound of antiplasmid effect having the general formula (I) 
characterised by that in the general formula
Q means: xcex2-picolinyl-methoiodide or 1-methyl-4-piperidyl or 3-(4xe2x80x2-fluorophenyl)-2,2-dimethyl-2-sila-propyloxy, or 2-dimethyl-amino-ethyl-methoiodide group and
Y means: 2,2-dimethyl-2-sila-hexyloxy or a 3-(4xe2x80x2-fluorophenyl)-2,2-dimethyl-2-sila-propyloxy or 5-(4xe2x80x2-fluorophenyl)-4,4-dimethyl-4-sila-pentyloxy or 3-trimethylsilil-propyloxy group.
The subject of the invention is the method for producing a hydroxy-benzoic acid derivative of the general formula (I) wherein Q and Y have the same meaning as defined above. The method is characterised by that
a) 4-hydroxy-benzoic acid ester is brought into reaction with such an organic halogen compound which provides a group Y as defined in the claim 1 under appropriate reaction circumstances,
b) the obtained 4-alkoxy-benzoic acid ester is hydrolysed into carboxylic acid,
c) the obtained 4-alkoxy-benzoic acid is transformed with a well-known method to acid chloride,
d) the obtained acid chloride is brought into reaction with such an amino-alcohol which provides a group Q as defined in the claim 1,
e) and the obtained amino-alcohol-ester is transformed into quaternary compound with methyl iodidexe2x80x94if necessary.
The subject matter of the invention is a pharmaceutical composition of antiplasmid effect characterised by that it contains the hydroxy-benzoic acid of the general formula where the meanings of Q and Y are the same as defined above, as an active substance.
The subject matter of the invention is also a method for producing a pharmaceutical composition characterised by that an effective quantity of the hydroxy-benzoic acid derivative of the general formula I wherein the meanings of Q and Y are the same as defined above is mixed with usual pharmaceutical ingredients.